Apr 08, 2024
Immunofluorescence staining protocol with Antigen Retrieval
- 1Van Andel Research Institute
Protocol Citation: madalynn.erb Erb 2024. Immunofluorescence staining protocol with Antigen Retrieval. protocols.io https://dx.doi.org/10.17504/protocols.io.bp2l62nk1gqe/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: February 09, 2024
Last Modified: May 31, 2024
Protocol Integer ID: 97924
Keywords: ASAPCRN
Abstract
This protocol details the immunofluorescence staining protocol with antigen retrieval.
Materials
ProLong™ Diamond Antifade MountantThermo FisherCatalog #P36961
Universal HIER antigen retrieval reagent (10X) AbcamCatalog #ab208572
Day 1
Day 1
Staining protocol for 35 µm free floating mouse brain sections.
Wash tissue sections 3 times (5 minutes each wash) in PBS to remove cryoprotectant solution.
Wash tissue sections for 00:05:00 in PBS to remove cryoprotectant solution (1/3).
5m
Wash tissue sections for 00:05:00 in PBS to remove cryoprotectant solution (2/3).
5m
Wash tissue sections for 00:05:00 in PBS to remove cryoprotectant solution (3/3).
5m
Mount tissue onto positively charged slides.
Allow slides to air dry then place in oven set at 37 °C -50 °C Overnight .
Note
Can use slide warmer or incubator for this step.
5m
Day 2
Day 2
If you would like to use a PAP pen to draw a box around your tissue sections, do that now and wait for slides to dry.
Wash slides in PBS for 00:05:00 .
5m
Preheat steamer with a Coplin jar containing the antigen retrieval buffer until temperature reaches 95 °C -100 °C (about 00:10:00
Note
This protocol uses Universal HIER antigen reagent as the buffer. This comes at 10X reagent, therefore, you need to dilute 1:10 with distilled water for 1x concentration of the desired volume.
10m
Immerse slides into the preheated buffer in the steamer for 00:30:00 .
30m
After 30 minutes turn off the steamer but allow to cool to Room temperature (about 20-00:30:00 ) before progressing to the next step.
30m
Wash slides in PBS 0.1% Trition X-100 (PBST) 2 x 2 min.
Wash slides in PBS 0.1% Trition X-100 (PBST) for 00:02:00 (1/2).
2m
Wash slides in PBS 0.1% Trition X-100 (PBST) for 00:02:00 (2/2).
2m
Wash slides in PBS 3 x 5 min.
Wash slides in PBS for 00:05:00 (1/3).
5m
Wash slides in PBS for 00:05:00 (2/3).
5m
Wash slides in PBS for 00:05:00 (3/3).
5m
Block for 02:00:00 at Room temperature in PBS (0.4% BSA, 10% Goat normal serum, 0.3% triton X-100).
Note
- Move slides to black slide box for this step and subsequent steps.
- Use 1-2ml solution for washes and 700 µL for antibody incubations.
2h
Incubate Overnight with primary antibody in PBS (2% Goat normal serum, 0.3% triton X-100) Overnight at Room temperature .
20m
Day 3
Day 3
9h
Wash slides in PBST 3 x 10 min.
Wash slides in PBST for 00:10:00 (1/3).
10m
Wash slides in PBST for 00:10:00 (2/3).
10m
Wash slides in PBST for 00:10:00 (3/3).
10m
Incubate slides with secondary antibody in PBS for 04:00:00 at Room temperature or at 4 °C Overnight .
8h
Wash slides in PBST 3 x 10 min.
Wash slides in PBST for 00:10:00 (1/3).
10m
Wash slides in PBST for 00:10:00 (2/3).
10m
Wash slides in PBST for 00:10:00 (3/3).
10m
Incubate slides with Hoescht stain (1:5000) for 00:30:00 at Room temperature .
30m
Wash slides in PBST 3 x 10 min.
Wash slides in PBST for 00:10:00 (1/3).
10m
Wash slides in PBST for 00:10:00 (2/3).
10m
Wash slides in PBST for 00:10:00 (3/3).
10m
Mount slides using ProLong™ Diamond Antifade Mountant.
Dry slides on a flat surface at Room temperature in the dark.
Note
Put them under aluminum foil or a box on the bench.
Store slides at 4 °C the next day.